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For each origin of replication, there are two "replication forks" at which new DNA is formed.
DNA replication: Directionality
5' → 3'
(3' hydroxyl at the end of the growing chain is a nucleophile which attacks the phosphorous on the nucleotide to be added; pyrophosphate eliminated and phosphodiester bond formed.)
Pol I (E. coli)
Single polypeptide chain polymerase. Repairing and "patching" DNA. Removes RNA primers.
Pol III (E. coli)
Made of 10 subunits; core enzyme responsible for DNA polymerization. "Clamps" on parent DNA and slides along it during replication. Main polymerizing enzyme in prokaryotes.
Short strand of RNA that first must be added to DNA to be replicated; polymerases can only add DNA nucleotides if this primer is present. (both prok. and euk.)
3' → 5' exonuclease activity
Proofreading function. Done one nucleotide at at time. All three polymerases (I, II, III) can do this.
5' → 3' exonuclease activity
Repair function. Done several nucleotides at a time; also how RNA primers are removed. Only Pol I can do this.
Single-strand binding protein
Stabilizes single-stranded DNA during replication by binding tightly; protects from hydrolysis by nucleases in the cell.
Pol I uses its 5' → 3' exonuclease activity to remove RNA primers or DNA mistakes missed by proofreading, then fills in behind with its polymerase activity.
Enzymes recognize that two bases are incorrectly paired, and remove mismatch (occurs when damage escapes normal exonuclease activity of Pol I and Pol III). Polymerases then fill in gaps. Enzymes know which is the parent strand because prokaryotes methylate DNA at certain locations.
Base excision repair
DNA glycolase removes base damaged by oxidation or chemical modification; AP endonuclease removes sugar and phosphate. Excision exonuclease removes more bases. Pol I fills in gap; DNA ligases seals phosphodiester backbone.
Common for DNA damage done by UV or chemicals. DNA removed by ABC excinuclease; DNA Pol I and DNA ligase fill in gap.
Complications in eukaryotic DNA replication vs. prokaryotic (3)
1. Multiple origins of replication
2. Timing must be controlled to that of cell division
3. More proteins and enzymes involved
The origins of replication (multiple) in eukaryotes. Specific DNA sequences between genes.
Origin recognition complex - multi-subunit protein that initiates replication. Bound to DNA thorugh cell cycle, but serves as attachment site for other proteins that control replication. (eukaryotes only)
Replication activator protein. First protein to bind to origin recognition complex. (eukaryotes only)
Replication licensing factors. Bind after replication activator protein. Some are cytosolic, so only have access to chromosome when nuclear membrane dissolves during mitosis. (eukaryotes only)
Pre-replication complex: Combination of DNA, origin recognition complex, replication activator protein, and replicatino licensing factors. (eukaryotes only)
Cyclin-CDK complex, and effects
Cyclins combine wit cyclin-dependent protein kinases and phosphorylate sites on RAP, RLFs, and ORC. Then RAP and RLFs dissociate and are degraded. (Thus, cyclin-CDKs initiate DNA replication AND prevent formation of another pre-RC.)
Eukaryotic DNA polymerases
α: adds ~20 nucleotides and is replaced by δ and ε.
δ: principal DNA polymerase in eukaryotes. Interacts with PCNA protein (proliferating cell nuclear antigen), which is the eukaryotic equivalent of the "sliding clamp" on the DNA.
ε: Involved in leading strand.
β: Repair enzyme.
γ: DNA replication in mitochondria.
Eukaryotic vs. prokaryotic polymerases
Separate exonucleotic enzymes exist in animal cells (in prokaryotes, the polymerases also act as exonucleases).
Primase activity in eukaryotic cells
Associated with Pol α (addition of RNA primer). Contrasts with primase enzyme in prokaryotes.
FEN-1 and RNase H1
Proteins that degrade RNA primer. Pol δ fills in gaps. DNA ligase seals nicks between fragments.
Proteins with which eukaryotic DNA is complexed. Histone biosynthesis occurs concurrently with DNA replication.
Prokaryotes vs. Eukaryotes: exonuclease activity
Prokaryotes: polymerases are ALSO exonucleases
Eukaryotes: Not all polymerases are exonucleases (there are separate enzymes to do this)
Prokaryotes vs. Eukaryoes: the origin of replication
Prokaryotes: ONE origin of replication
Eukaryotes: MULTIPLE origins of replication
Prokaryotes vs. Eukaryotes: Okazaki fragments
Prokaryotes: 1000-2000 residues long
Eukaryotes: 150-200 residues long
Prokaryotes vs. Eukaryotes: DNA complex
Prokaryotes: No DNA/protein complex
Eukaryotes: Have histones complexed to DNA
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